Method of targeting a specific location in a body

ABSTRACT

Micellular particles such as small unilamellar vesicles of less than 2000 Å loaded with  111  In are administered to BALB/c mice in which EMT6 tumors had been induced. Whole body scintographs of the mice to which either neutral or positively or negatively charged vesicles had been administered show a substantial quantity of the vesicle entrapped  111  In localized in the tumor. Blocking of macrophages in the liver and spleen by first administering unlabeled, aminomannose substituted vesicles before administration of the labeled vesicles increases uptake of the  111  In labeled vesicles in the tumor.

BACKGROUND OF THE INVENTION RELATIONSHIP TO OTHER APPLICATIONS

This application is a continuation-in-part of application, Ser. No.363,593, filed Mar. 30, 1982, now abandoned "Method of Targeting aSpecific Location in a Body".

FIELD OF THE INVENTION

This invention relates to a method of targeting specific locations as,for example, tumors, in a body, by use of micellular particles such asphospholipid vesicles. The invention may be used for diagnosis and/ortreatment of such abnormalities.

DESCRIPTION OF PRIOR ART

Before various abnormalities in a patient's body can be diagnosed andtreated, it is often necessary to locate the abnormalities. This isparticularly true of abnormalities such as malignant tumors since thetreatment of such tumors is often on a localized basis. For example, thelocation of cancer cells has to be identified so that a therapeuticagent can be directed to such cells to eliminate the tumor.

Various attempts have been made over an extended number of years toidentify specific locations, such as tumors, in a patient's body bysimple techniques. For example, it would be desirable to identify thelocation of cancer cells by a simple method involving the introductionof a particular chemical to the patient's body and the movement of suchchemical to such specific locations. It would also be desirable to treatthe cancer by introducing modified chemicals into the patient's body andhaving such chemicals move to specific locations to combat the cancercells at such locations. In spite of such attempts, however, simpledelivery systems for targeting specific locations, such as tumors, fortreatment or diagnosis do not exist as yet.

Placing a chemotherapeutic drug in the body orally, subcutaneously orintravenously can result in harm to the normal cells in the body whichtake up the drug and a worsening in the patient's condition, withoutachieving the desired reduction in tumor cell activity. In the past,this toxicity to normal cells in the patient's body has been a majordisadvantage in the treatment of tumors with chemotherapeutic agents.The lack of efficacy of such chemotherapy is also attributable to thefailure of the freely circulating drug to localize within tumor cellsbefore it is excreted or taken up by other cells in the body.

Prior attempts to improve treatment of tumors by chemotherapeutic agentshave included encapsulation of such agents within biodegradablephospholipid micellular particles in the form of vesicles or liposomes.Encapsulation is thought to reduce the potential toxicity from thecirculating drugs. Researchers have also sought to utilize suchencapsulation to selectively target tumors within a body for delivery ofchemotherapeutics. However, until the invention disclosed in the presentapplication and the related application Ser. No. 363,593, efforts tolocate or treat tumor cells with drug-encapsulating targeting particleshave not been successful.

The inability to provide a satisfactory targeting method is believed tobe due to the nature of the solid tumors and their metastases which arelocated in extravascular tissues. Thus, to accomplish targeting ofintravenously injected radiolabelled or chemotherapeutic agents to thetumor cells, the agents must leave the normal circulation by crossingthe blood vessel membranes to enter the extravascular tissues. Thismovement is known as "extravasation". In addition the encapsulated agentmust cross the tumor cell membrane. Normally, small substances such assmall molecular weight proteins and membrane-soluble molecules can crosscell membranes by a process known as passive diffusion. However, passivediffusion will not allow sufficient accumulation of larger particlescarrying drugs within cells to reach therapeutic levels. Additionally,cells can actively transport materials across the membrane by a processsuch as pinocytosis wherein extracellular particles are engulfed by themembrane and released inside the cell. Entry of encapsulating particlesinto individual cells may occur by pinocytosis.

Progress in targeting such specific locations with chemotherapeuticdrugs has been hampered by the inability to accomplish and detectmovement of drug carriers across blood vessel membranes. In the usualcase, large structures such as drug encapsulating vesicles cannot escapefrom blood vessels such as capillaries, and thus remain in circulation.

An understanding of extravasation, however, requires an examination ofthe structure of the vascular morphology of a tumor. Various bloodvessels are associated with tumors, in particular capillaries. It is nowknown that tumor capillaries may exhibit alterations in their structure,such as fenestrations, as a result of tumor cell growth patterns. H.I.Peterson, Vascular and Extravascular Spaces in Tumors: Tumor VascularPermeability, Chapter III, Tumor Blood Circulation, H. I. Peterson, Ed.(1979). Studies of tumor capillary permeability reveal morphologicvariations in the capillaries which allow some substances to cross thecapillary membrane. Such variations include defects in vascularendothelium from poor cell differentiation, or breaks in vascular wallsas a result of invading tumor cells. H.I. Peterson, supra.

Notwithstanding such knowledge of tumor vascular morphology, researcherssuch as Peterson have concluded that transport of large molecules ormaterials across the tumor capillary wall occurs as a result of passivediffusion and that "concentrations of active drugs sufficient fortherapeutic effect are difficult to reach." H. I. Peterson, supra, at83.

Prior to such morphologic studies, early reports suggested that vesiclesmight undergo transcapillary passage across the capillary membranes intotumor cells. G. Gregoriadis, Liposomes in Biological Systems,Gregoriadis, Ed., Ch 2, (1980). However, available data indicated thatthe vesicles were unstable in vivo and that the radiolabel may haveleaked, thus apparently prompting alternative theories such as longercirculation of vesicles in the blood with release of drugs at a slowerrate and interaction of the liposomes with the capillary walls withoutcrossing the wall surface, which presumably resulted in the drugs withintumors. Id. Other researchers simply have concluded that the vesicles donot penetrate vascular walls after intravenous administration. B. Rymanet al., Biol. Cell, Vol 47, pp. 71-80 (1983); G. Poste, Biol. Cell, Vol.47, pp. 19-38 (1983).

Thus, although the prior art has recognized that vesicles carryingtherapeutic drugs must cross vascular barriers to reach tumor cells, theexperience of the art has taught that intravenous administration is noteffective to deliver encapsulated drugs to extravascular tumor cells.This invention accordingly provides simple methods of enhancingextravasation of encapsulated chemotherapeutic agents to tumor cellswithin a body. The method of this invention further provides for theidentification of such tumor sites in the body.

SUMMARY OF THE INVENTION

The method of this invention includes the provision of phospholipidmicellular particles such as vesicles. Pure (more than approximately 98%pure) neutral phospholipid molecules are incorporated into small (lessthan 2000Å) micelles so that they are a component of external surface.The phospholipid molecules and/or vesicle contents may be radiolabeledto enhance the identity of the specific location and the diagnosis ofthe tumor at the specific location.

The phospholipid molecules may constitute distearoylphosphatidylcholine. The stability of the distearoyl phosphatidylcholinemicelles may be enhanced by the incorporation of cholesterol. Positivelycharged molecules such as stearylamine or aminomannose or aminomannitolderivatives of cholesterol or negatively charged molecules such asdicetyl phosphate may also be incorporated into the vesicles.

When phospholipid micelles are introduced into the blood stream of apatient, the micelles move to the specific locations of cancerous growthin the patient's body, which may then be identified and treated. Drugsmay be included in phospholipid vesicles and such drug-bearing vesiclesmay then be introduced into the patient's body for targeting the tumorlocations.

To enhance movement of the phospholipid vesicles to the specificlocations, positively charged phospholipid vesicles may first beintroduced into the patient's blood stream to block the macrophages inthe patient's body. The positively charged molecules bound to suchphospholipid vesicles may be an aminomannose or aminomannitol derivativeof cholesterol. Concurrently or after a suitable period of time such asapproximately one (1) hour, other phospholipid vesicles may beintroduced into the patient's blood stream to move to the specificlocations in the body. Such phospholipid vesicles may includecholesterol and may be neutral or may be positively charged as by theinclusion of a stearylamine or aminomannose or aminomannitol derivativeof cholesterol or may be negatively charged as by the inclusion of adicetyl phosphate.

When the phospholipid vesicles are introduced into the body to targettumors, indium-111 may be used as the labelling agent. The indium-111may be chelated to a suitable material such as nitrilotriacetic acid(NTA). NTA is advantageous because it forms a weak bond with theindium-111. As a result, when the phospholipid vesicles reach the tumor,the nitrilotriacetic acid is displaced by proteins at the tumor. Sincethe proteins have a strong bond with the indium-111, the indium-111remains at the tumor for a long period of time (in excess of 24 hours),which provides for easy identification of the tumor over the extendedperiod of time.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a table illustrating the targeting of phospholipid vesicles totumors in a body;

FIG. 2 is a table illustrating the targeting and blocking of macrophagesin the liver and spleen by phospholipid vesicles;

FIG. 3 is a table illustrating the targeting of phospholipid vesicles totumors in the body after the blocking of the macrophages in the liverand spleen; and

FIG. 4 is a table illustrating the enhanced delivery of drugs to a tumorin a body by the use of phospholipid vesicles.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, "micellular particle" and "micelles" refer to particleswhich result from aggregations of amphiphilic molecules. In thisinvention preferred amphiphiles are biological lipids. Micelles arewater-soluble aggregates of molecules with hydrophobic and hydrophilicportions (so-called amphiphilic molecules) which associatespontaneously. Such micelles can be in the form of small spheres,ellipsoids or long cylinders, and can also consist of bilayers with twoparallel layers of amphiphilic molecules. Such bilayered micellesusually take the shape of spherical vesicles with an internal aqueouscompartment. Useful compositions of these micelles include phospholipidmolecules in the structure.

"Vesicle" refers to a micelle which is in a generally spherical form,often obtained from a lipid which forms a bilayered membrane and isreferred to as a "liposome". Methods for forming these vesicles are, bynow, well known in the art. Typically, they are prepared from aphospholipid, for example, distearoyl phosphatidycholine, and mayinclude other materials such as neutral lipids, for example,cholesterol, and also surface modifiers such as positively or negativelycharged compounds.

Materials and Methods

Liposome Preparation. Small unilamellar vesicles (SUV) with theionophore A23187 were prepared from distearoyl phosphatidycholine(DSPC), cholesterol (Ch), dicetyl phosphate (DP), stearylamine (SA) andthe 6-aminomannose (AM), and 6-aminomannitol (AML) derivatives ofcholesterol, according to previous methods. Briefly, chloroformsolutions of 10 mg lipid with the following molar ratios: DSPC:Ch, 2:1;DSPC:Ch:X, 4:1:1 where X=SA, DC or AML; and DSPC:Ch:AM, 8:3:1, wereevaporated to dryness under nitrogen (N₂,) and further dried undervacuum overnight. Each tube was filled with 0.6 ml phosphate 10 mMphosphate buffered 0.9 saline, pH 7.4(PBS), containing 1 mMnitrilotriacetic acid (NTA) and sonicated under N₂, for 5 to 15 minuteswith a MSE sonicator equipped with a titanium microtip.

Liposomes were annealed at 60° C. for 10 minutes and centrifuged at300×g. Liposomes were separated from unencapsulated NTA with a 30×1.5 cmSephadex G-50 column. Liposome size was determined by electronmicroscopy of preparations negatively stained with uranyl acetate. Allvesicle types were shown by electron microscopy to have a mean diameterless than 0.1 microns (1000Å). For example, DSPC:Ch vesicles had a meandiameter of approximately 528Å. However, vesicles as large asapproximately 2000 Angstroms are believed to be satisfactory inobtaining the desired results of this invention, although the preferredrange is approximately 500 to about 700Å.

The vesicles obtained as described above are chemically pure. By"chemically pure" is meant that the materials which constitutephospholipid vesicles are more than 98% pure. For example, when thephospholipid chemical added is distearoyl phosphatidylcholine, thismaterial is used at more than 98% purity. The same constraint holds forother components, such as cholesterol, which compose the vesicle. Thephospholipid vesicles obtained as described above are stable wheninjected into experimental animals.

The aminomannose and aminomannitol derivatives of cholesterol extendexternally from the phospholipid particles. Thus, when such derivativesare incorporated or associated into the surfaces of vesicles or othermicelles, an amine moiety is provided that extends approximately 5-15Angstroms, preferably about 10 Angstroms, beyond the surface of themicelles. In the case of vesicles, it appears that the appropriatemolecular design comprises a hydrophobic portion which serves to anchorthe molecule within the vesicular bilayer, and a linking portion whichis at least mildly hydrophilic which spans the requisite distancebetween the hydrophobic region and the amino functional group. Thehydrophilicity is apparently required to prevent the link frominternalizing within the bilayer also and thus serves to "extend" theamine from the surface. An example of a successful extended amine withinthe context of this invention is a 6-aminomannose cholesterol derivativesuch as, for example,6-(5-cholesten-3β-yloxy)hexyl-6-amino-6-deoxyl-thio-D-mannopyranoside.In this example, the cholesterol portion provides the hydrophobicmoiety, while the aminomannose is relatively hydrophilic. Otherembodiments are also possible: other amino sugars attached to othercholesterol derivatives, for example, are equally suitable asalternative embodiments of the hydrophilic and hydrophobic portions.Polyamines and polyamino acids which can be bound covalently orassociated by other means to the vesicle or other micelle surface mayalso be used.

The amino derivatives and cholesterol tend to impart stability to thephospholipid vesicles. Cholesterol may be included in the range ofapproximately 0% to 50% of cholesterol by weight and the remainderconstituting the phospholipids. The charged molecules such as thestearylamine, the dicetyl phosphate and the aminomannose andaminomannitol derivatives of cholesterol may be included in the range of0% to 20% by weight of the charged molecules and the remainderconstituting the phospholipids.

The chemically pure liposome compositons discussed above are quitestable to leakage in vitro and in vivo. However, phospholipid mixturessuch as egg lecithin form more fluid membranes than pure phospholipids.As a result, liposomes from natural lecithin mixtures are less stable toleakage of their contents than pure phopholipids.

In-111 Loading Procedure. Loading of In-111 into preformed liposomes wasfacilitated by the presence of A23187 in the lipid bilayer. In-111 wasloaded into liposomes at 60°-80° C. as previously described. Incubationswere terminated by the addition of 10mM ethylenediaminetetraacetic acid(EDTA) in 10 mM phosphate buffered 0.9% sodium chloride, pH 7.4 (PBS),and free In-111 was separated from the loaded liposomes bychromatography on Sephadex G-50. Up to 90% of the added In-111 could beincorporated into preformed liposomes by this technique, and specificactivities of up to 300 μCi/mg lipid have been obtained.

EMT6 Tumor Growth. Male BALB/c mice weighing 20-25 g were injectedsubcutaneously on the right hind leg with 5×10 EMT6 cells in 0.1 mlsterile phosphate buffered saline. Tumors were allowed to grow for 10-20days prior to using these animals for imaging studies. At this stage,tumors weighed between 0.2 and 0.4 gm. Up to 0.5 ml PBS containing 1 to2 mg liposomes loaded with up to 30 μCi in-111 were injected into thetail vein of each animal. Control animals were injected with In-111-NTAwhich had not been encapsulated in liposomes.

Gamma Camera Imaging. At one (1) hour and at twenty-four (24) hoursafter injecting In-111 loaded liposomes, each animal was anesthetizedwith 40 mg/kg sodium pentobarbital and positioned on a platform 12 cmfrom the gamma scintillation camera equipped with a 6 mm pinhole.Whole-body dorsal images were acquired on x-ray film and correspondingdigitized data were stored on magnetic discs for computer analysis.

Biodistribution of Radioactivity. Immediately after the twenty-four (24)hour images were acquired, animals were sacrificed and dissected todetermine the organ distribution of radioactivity. Organs or tissueswere excised, washed in PBS, blotted dry, and weighed. Radioactivity wasmeasured in a well-type gamma-ray spectrometer and quantitated based onactivity present in liposomes before injection. In some experiments, thegamma-ray perturbed angular correlation technique was used to measurethe rotational correlation time of the In-111 in individual tissues andthereby assess the proportion of isotope remaining in intact liposomes.

RESULTS

Whole body scintographs were made of tumor bearing mice which had beeninjected intravenously with In-111 NTA small phospholipid vesicles SUV24 hr previously. EMT6 tumor images were clearly discernible in animalsinjected with neutral, negative and positively charged phospholipidvesicles. A comparison of the biodistribution of In-111 NTA delivery byeach of these vesicle types can be made from the data presented inFIG. 1. As will be seen from the second column of FIG. 1, neutralphospholipid vesicles provided the best delivery of In-111 to tumortissue. The specific targeting of the phospholipid vesicles to thetumors in this instance was at least as high as the targeting of thephospholipid vesicles to the liver or spleen, the usual target tissuesof liposomes, and was nearly 8 times greater than the specific activityobserved at the tumors when free In-111 NTA was injected in vivo. Thiswill be seen from a comparison of the results shown in the first andsecond columns of FIG. 1. It can also be seen in FIG. 1 that, as liverand spleen uptake of In-111 decreases, the concentration of thephospholipid vesicles remaining in the blood increases. Also theincrease in tumor associated radioactivity correlates approximately withthe blood level of In-111.

Applicants have previously demonstrated a strong association with EMT6tumor cells in vitro of liposomes with 6-aminomannose derivatives ofcholesterol. Applicants accordingly attempted tumor imaging withphospholipid vesicles of aminomannose derivatives of cholesterol wheresuch vesicles were labeled with in-111. Applicant's observations in thisexperiment confirmed that the vast majority of In-111 in suchphospholipid vesicles ultimately is deposited in the liver and spleen.Tumor images could not be obtained with such phospholipid vesicles asdemonstrated in columns 2 and 3 of FIG. 2 by the low deposition of thephospholipid vesicles in the tumor. The low deposition of thephospholipid vesicles in the tumor may result from the fact that most ofsuch vesicles are taken up by the liver and spleen.

Liposomes with a lower concentration of the 6-AM derivative ofcholesterol do not get trapped in the lung, so it seemed reasonable toassume that AM/2 vesicles (third column of FIG. 2) loaded with In-111might be better tumor imaging agents than the material shown in thesecond column of FIG. 2. A comparison of the second and third columns ofFIG. 2 shows that this was not the case. In fact, the AM/2 vesicles hada very high affinity for the liver and spleen. For example, after aperiod of 24 hours from the time of injection of the lipid vesicles inthe blood stream, the combined radioactivity in the liver and spleenaveraged greater than 75% of the total injected dose. This was thehighest amount of liver and spleen uptake of vesicles observed of theseveral lipid composition studies.

Applicants have previously shown that positively charged liposomes werebound to EMT6 cells in vitro to a much greater extent than eitherneutral or negatively charged liposomes. Applicants accordinglyinvestigated AML derivatives of cholesterol, another syntheticglycolipid derivative with positive charge. These AML liposomes did showa lower affinity for liver and spleen (Column 4 of FIG. 2) and aslightly increased uptake by tumor compared to that provided by AM/2liposomes (Column 2 of FIG. 2). However, this level of tumor-associatedradioactivity was still three to ten times less than observed in sheexperiment with neutral, positive and negative liposomes as shown inFIG. 1.

In further experiments, applicants injected mice with either a salinesolution or with 8 mg AM/2 liposomes. The saline solution provided acontrol and did not block the macrophages in the liver and spleen in themanner discussed above. This is shown in FIG. 3. The AM/2 liposomesprovided a positive charge and were effective in blocking themacrophages in the liver and spleen. This is also shown in FIG. 3. Sincethe macrophages in the liver and spleen were blocked, any subsequentinjection of phospholipid vesicles into the blood stream of the body hadan increased opportunity to become targeted to the tumor.

One hour after the injection of the liposomes as discussed in theprevious paragraph, 1 mg of the type of liposomes discussed above inrelation to FIG. 1 was injected in the mice. These liposomes containedIn-111. Twenty-four (24) hours afterwards, mice were sacrificed anddissected to determine biodistribution of In-111.

FIG. 1 indicates the amount of In-111 targeted to the different parts ofthe body when phospholipid vesicles containing In-111 are introducedinto the blood stream without any previous blockade of the macrophagesin the liver and spleen. In contrast, FIG. 3 indicates the amount ofIn-111 targeted to the different parts of the body when phospholipidvesicles containing In-111 are introduced into the blood stream after aprevious blockade of the macrophages in the liver and spleen. As will beseen, the amount of the In-111 targeted to the tumor significantlyincreased in most instances in FIG. 3 for the individual phospholipidvesicles than for the corresponding phospholipid vesicles in FIG. 1.Furthermore, the amount of the In-111 received at the liver and spleenin FIG. 3 is significantly reduced from the amount of the In-111received at the liver and spleen in FIG. 1.

As will be seen from a comparison of FIGS. 1 and 3, a significant amountof the-phospholipid vesicles is targeted to the tumor even when themacrophages in the liver and spleen are not previously blocked. However,the amount of phospholipid vesicles targeted to the tumor issubstantially increased when the macrophages in the liver and spleen areblocked before the phospholipid vesicles to be targeted to the tumor areintroduced into the body.

In the experiments discussed above, the phospholipid vesicles to betargeted to the tumor were introduced into the blood streamapproximately one (1) hour after the introduction of the phospholipidvesicles into the bloodstream to block the macrophages it, the liver andspleen. It will be appreciated, however, that other time periods mayalso be used, including time periods considerably shorter than one (1)hour. Since the phospholipid vesicles blocking the liver and spleen areeffective for an extended period, the introduction of the phospholipidvesicles to target the tumor may be considered as concurrent with theintroduction of the phospholipid vesicles to block the liver and spleen.

As previously described, neutral DSPC:Ch phospholipid vesicles deliverIn-111 to EMT6 murine tumors in sufficient quantity to allow definitivelocalization of tumor by gamma camera imaging. This tumor-associatedspecific radioactivity (% dose/gram tissue) is equal to levels achievedin liver and spleen, a finding which was not previously observed byothers employing liposomes as tumor imaging agents.

There are several improvements in liposome technology employed in thepresent invention which may explain why better tumor imaging is achievedthan has been previously observed by others. One such improvement isthat applicants have loaded In-111 into preformed liposomes. By thishighly efficient method, specific activities of 200-300 μCi In-lll/mglipid have been obtained. Another improvement is that applicants haveused highly purified phospholipid vesicles as discussed above.

A further improvement has been that In-111 has been encapsulated in theNTA complex. NTA is a relatively weak chelator and, in the presence ofserum, NTA is displaced. Thus, when the phopholipid micelles containingthe In-111 are targeted to the tumor, the NTA becomes displaced byprotein at the tumor. The In-111 becomes tightly associated with theprotein at the tumor. Since this protein is within a cell, the In-111 isfixed at the position of the tumor. This circumstance provides twodistinct advantages for the purpose of imaging. The first is that littleradioactivity is lost due to leakage. After correcting for decay,applicants typically observed that 90% of the initial radioactivityremained in the animal at least twenty-four (24) hours after injection,based on the times required to accumulate a fixed number of counts withgamma counter. A second advantage is that when a label such as In-111remains fixed at the site of liposome destruction, one can obtaininformation on rate, as well as total amount, of liposome uptake by thetissue.

Thus, the high tumor specific activities observed in this study are theresult of a continuous accumulation of In-111 within the tumor over atwenty-four (24) hour period. By comparison, EDTA contained withinstearylamine vesicles forms a strong chelate in comparison to NTA. EDTAis not displaced at the tumor by proteins. Thus, the In-111 will notremain fixed within the cell. For example, when EDTA was chelated toIn-111 in a phospholipid vesicle, only 25% of tumor specific activitywas achieved, compared to In-111 NTA loaded liposomes.

The phospholipid vesicles may be used to provide an enhanced delivery ofdrugs or radionuclides to tumors in the body. This may be seen from theresults of experiments specified in the table constituting FIG. 4. Inthese experiments [³ H] Methotrexate (MTX) was injected directly intotumor-bearing mice as a control. The amount of the [³ H]MTX is directedto the tumors after a period of four (4) hours is illustrated in the rowdesignated in FIG. 4 as "Free [³ H]MTX".

Phospholipid vesicles containing DSPC:Ch:SA in the ratio of 4:1:1 werelabeled with [¹⁴ C] cholesteryl oleate and the [³ H]MTX was entrapped inthe phospholipid vesicles. As will be seen, the amount of thephospholipid vesicles targeted to the tumors is almost three (3) timesgreater than the amount of the free MTX directed to the tumor.

The liver and spleen were also blocked in the manner described above andshown in FIG. 2 before the phospholipid vesicles containing DSPC:Ch:SA,as described in the previous paragraph, were targeted to the tumors. Thelast column of the table in FIG. 4 illustrates the targeting of thesephospholipid vesicles to the tumors after the blocking of the liver andspleen. As will be seen, the amount of the phospholipid vesiclestargeted to the tumors under such circumstances was almost the same asthe amount discussed in the previous paragraph.

As indicated previously, the micellular particles are to be less than2000Å in diameter, preferably in the 500 to approximately 700Å range. Todemonstrate further the significance of such size limitation, Table Ishows biodistribution data from four mice that received REV vesiclescontaining In-111 NTA. The larger vesicles were prepared by reversedphase evaporation following the method of Syoka and Papahadjopoulos,Proc. Natl. Acad. Sci. USA, 75: 4194-4198 (1978), and were approximatelyfive times larger than SUV's, but with identical chemical composition.To maximize tumor accumulation of the radiolabel agent, the animals weresacrificed when the mean lesion size was only 4±1 mg. Such structures,approximately 2 millimeters in largest dimension, were within the limitsof the dissection technique. Uptake of REV's by the malignancies wasonly 2.1±0.1% ID/g. This value was somewhat above blood levels, but onetenth or less than that found in comparably sized Lewis Lung Carcinomasusing SUV's (Table 2). In order to establish that tumor size effects arenot different for the larger vesicles, two additional mice were injectedwith REV's when their carcinomas were considerably larger. Tumoraccumulations were 1.5 and 1.2% ID/g in 320 and 2.10 mg lesionsrespectively. Thus, larger vesicles of DSPC:Chol have very significantlyreduced accumulation in Lewis Lung Carcinoma (p<0.005 using Tables 1 and2).

It has also been found that tumor uptake of encapsulated agent decreasessignificantly with increasing tumor size. For LLC lesions between 0.1and 0.5 g, uptake varied between 25 and 12% ID/g. Very small metastases(4 and 8 mg) found in the lung after s.c. implantation had uptake ofapproximately 50% ID/g. For larger lesions, a slow decrease in tumoraccumulation was observed out to 1.8 g where the value approached some10% ID/g. Most of the variation with size occurred in the range 0.02 to0.2 g; i.e. between 0.1 and 1.0% of the animal's total mass. It wasobserved during dissection that the larger tumors had relativelyenhanced necrotic zones, which explains, at least in part, thisdependence upon size.

Although this invention has been described and illustrated withreference to particular applications, the principles involved aresusceptible of numerous other applications which will be apparent topersons skilled in the art. The invention is, therefore, to be limitedonly as indicated by the scope of the appended claims.

                  TABLE 1                                                         ______________________________________                                        BIODISTRIBUTION OF REVERSED PHASE                                             EVAPORATION VESICLES (REV) IN MICE WITH                                       LEWIS LUNG CARCINOMA AND GRANULOMA                                                           % injected dose per                                            Tissue         per gram of tissue*                                            ______________________________________                                        Blood          1.1 ± 0.2                                                   Tibias         5.5 ± 0.5                                                   Lung           3.7 ± 0.8                                                   Liver          50.7 ± 2.1                                                  S & L Intestine#                                                                             0.9 ± 0.0                                                   Kidney         5.9 ± 0.3                                                   Spleen         73.2 ± 13.9                                                 Carcass        0.9 ± 0.1                                                   Stomach#       0.2 ± 0.0                                                   Muscle         0.3 ± 0.0                                                   Skin           0.8 ± 0.0                                                   S.C. LLC       2.1 ± 0.1                                                   Tumor                                                                         Granuloma      0.6 ± 0.1                                                   % of Recovery  81.8 ± 0.9                                                  Tumor Mass (mg)                                                                              4 ± 1                                                       ______________________________________                                         *Mean values ± standard error of the mean                                  N = number of mice per group                                                  # values include organ contents                                          

                  TABLE 2                                                         ______________________________________                                        TISSUE DISTRIBUTION OF                                                        VESICLE-ENCAPSULATED IN-111-NTA                                               IN MICE WITH SUBCUTANEOUSLY IMPLANTED                                         LEWIS LUNG CARCINOMA                                                                  24 h % Injected Dose Per Gram of Tissue*                                      Number of days after subcutaneous                                             implantation of Lewis Lung Carcinoma                                            8           11          17                                          Tissue    N = 4+      N = 4+      N = 4+                                      ______________________________________                                        Blood     8.1 ± 0.1                                                                              11.4 ± 2.3                                                                             1.3 ± 0.4                                Tibias    6.4 ± 0.6                                                                              6.8 ± 0.6                                                                              4.7 ± 0.7                                Lung      5.7 ± 0.5                                                                              15.1 ± 1.4                                                                             12.6 ± 1.6                               Liver     50.0 ± 2.4                                                                             50.5 ± 1.9                                                                             36.1 ± 3.2                               S & L     4.3 ± 0.2                                                                              4.9 ± 0.6                                                                              1.8 ± 0.3                                Intestine#                                                                    Kidney    15.1 ± 0.5                                                                             15.8 ± 0.4                                                                             9.2 ± 0.2                                Spleen    57.0 ± 5.6                                                                             50.0 ± 6.7                                                                             22.2 ± 3.2                               Carcass   2.3 ± 0.1                                                                              2.8 ±  0.2                                                                             1.6 ± 0.1                                Stomach*  3.5 ± 0.6                                                                              4.3 ± 1.3                                                                              1.5 ± 0.5                                Muscle    1.1 ± 0.1                                                                              0.8 ± 0.0                                                                              0.4 ± 0.0                                Skin      5.6 ± 2.7                                                                              2.1 ± 0.3                                                                              2.4 ± 0.2                                s.c. LLC  23.7 ± 2.7                                                                             17.0 ± 3.3                                                                             9.8 ± 0.9                                Tumor                                                                         % Recovery                                                                              102.8 ± 0.6                                                                            105.0 ± 1.9                                                                            91.3 ± 1.5                               Tumor mass (g)                                                                          0.13 ± 0.04                                                                            0.16 ± 0.04                                                                            1.67 ± 0.39                              ______________________________________                                         *Mean values ± standard error of the mean                                  +N = number of mice per group                                                 # Values include organ contents                                          

We claim
 1. A method of targeting a tumor in a body with an agent forthe diagnosis or treatment therefor comprisinga) providing micellularparticles of less than 2000 Å comprising chemically pure phospholipidmolecules; b) incorporating the agent for diagnosis or treatment intothe particles; and c) introducing the micellular particles into thebloodstream of the body to obtain movement of the particles to thetumor.
 2. A process according to claim 1 wherein the agent is aradioactive element.
 3. A process according to claim 2 wherein the agentemits gamma radiation.
 4. A process according to claim 3 wherein theagent comprises ¹¹¹ In.
 5. A method as set forth in claim 1 wherein theparticles constitute distearoyl phosphatidylcholine.
 6. A method as setforth in claim 5 wherein cholesterol is included in the phospholipidparticles to enhance the stability of the particles.
 7. A method setforth in claim 1 wherein charged molecules are also attached to thevesicles.
 8. A method as set forth in claim 7 wherein the small,chemically pure phospholipid vesicles are neutral and wherein thecharged molecules are positively charged or negatively charged.
 9. Amethod of targeting a tumor in a body with an agent for the treatment ordiagnosis therefor, comprising:a) providing small vesicles of less than2000 Å comprising chemically pure phospholipid vesicles; b) bindingcholesterol to such phospholipid vesicles c) binding charged moleculesto such phospholipid vesicles in the range of approximately 0% to 20% byweight of the charged molecules and the remainder constituting thephospholipids; d) incorporating the agent for diagnosis or treatmentinto the vesicles and e) introducing such phospholipid vesicles into thebloodstream of the body to obtain the movement of such phospholipidvesicles to the tumor in the body.
 10. A method of targeting a tumor ina body with an agent for the diagnosis or treatment thereof comprisingthe steps of:a) providing vesicles comprising chemically purephospholipid molecules having positively charged amino groupsincorporated therewith; b) introducing such positively charged vesiclesto the bloodstream of the body to block macrophages in the body; c)providing small vesicles of less than 2000 Å comprising chemically purephospholipid molecules having incorporated therein the agent fordiagnosis Dr treatment; and d) introducing the small vesicles with agentinto the bloodstream of the body subsequent to the blocking ofmacrophages to obtain movement of the small vesicles to the tumor.
 11. Amethod as set forth in claim 10 wherein charged molecules are alsoincorporated into said small vesicles and wherein the charged moleculesmay be positively charged and may constitute stearylamine, oraminomannose or aminomannitol derivatives of cholesterol, or may benegatively charged and may constitute dicetyl phosphate and wherein thecharged molecules extend externally from the phospholipid vesicles. 12.A method as set forth in claim 11 wherein the phospholipid constitutesdistearoyl phosphatidyl choline.
 13. A method as set forth in claim 10wherein the charged molecules are selected from a group consisting ofstearylamine, aminomannose or aminomannitol derivatives of cholesteroland dicetyl phosphate.
 14. A method according to claim 10 wherein theagent is a label which can be detected in vivo and further comprisingthe step of determining the location of the small vesicles in the bodyby detecting said label.
 15. A method of targeting tumors in a body,comprising the steps of:a) providing small vesicles of less than 2000 Åcomprising neutral phospholipids; b) adding cholesterol to such neutralphospholipid vesicles; c) incorporating charged molecules into suchneutral phospholipid vesicles in the range of approximately 0% to 20% byweight of the charged molecules and the remainder constituting thephospholipids. d) incorporating a drug into such phospholipid vesicles;and e) introducing such phospholipid vesicles into the body to obtainthe targeting of such phospholipid vesicles to the specific locations ofthe tumor in the body.
 16. A method of targeting tumors in a body,including the steps of:a) providing small vesicles of less than 2000 Åcomprising chemically pure phospholipid molecules; b) modifying aportion of the phospholipid vesicles to provide for the blockage of themacrophages in the body by such modified phospholipid vesicles; c)initially introducing the modified phospholipid vesicles to thebloodstream of the body to block uptake by the macrophages in the body;d) incorporating an agent for diagnosis or treatment of the tumor in asecond portion of the vesicles; and e) subsequently introducing thesecond portion of phospholipid vesicles to the bloodstream of the bodyto obtain a movement of lipid vesicles and agent to the tumor in thebody.
 17. A method of targeting tumors in the body, including the stepsof:a) providing small, chemically pure phospholipid vesicles of lessthan 2000 Å ; b) incorporating monosaccharide derivatives of cholesterolinto the phospholipid vesicles so that the monosaccharides extendexternally from the vesicles; c) incorporating into the vesicles anagent for the diagnosis or treatment of the tumor; and d) introducingthe monosaccharide-containing lipid vesicles into the bloodstream toobtain the movement of said vesicles to the location of the tumor in thebody.
 18. A method as set forth in claim 17 wherein the phospholipidconstitutes distearoyl phosphatidyl choline.
 19. A method as set forthin claim 18, including the step of blocking the macrophages in the bodybefore introducing said phospholipid vesicles into the body.
 20. Amethod as set forth in claim 9 wherein the phospholipids in thephospholipid vesicles constitute distearoyl phosphatidylcholine andwherein the charges in the charged phospholipid vesicles are provided byaminomannose or aminomannitol derivatives of the cholesterol and arecoupled to the phospholipid vesicles.
 21. A method as set forth in claim20 wherein the charged molecules extend externally of the phospholipidvesicles.
 22. A method as set forth in claim 21 wherein the chargedmolecules are selected from the group consisting of stearylamine,dicetylphosphate, and aminomannose and aminomannitol derivatives ofcholesterol.
 23. A method as set forth in claim 16 wherein the modifiedphospholipid vesicles are charged.
 24. A method as set forth in claim 16wherein cholesterol is coupled to the phospholipid vesicles.
 25. Amethod as set forth in claim 24 wherein aminomannose or aminomannitolderivatives of cholesterol are coupled to the phospholipid vesicles tomodify the phospholipid vesicles and wherein the phospholipid vesiclesare charged by a material selected from the group consisting ofstearylamine, dicetyl phosphate and aminomannose and aminomannitolderivatives of cholesterol.
 26. A method as set forth in claim 25wherein charged molecules in the range of 0% to 20% are coupled to thephospholipid vesicles to modify the phospholipid vesicles for blockingthe uptake of other phospholipid vesicles by the macrophages in the bodyand the charged molecules extend externally from the phospholipidvesicles.
 27. A method as set forth in claim 26 wherein thephospholipids constitute distearoyl phosphatidylcholine.
 28. A method asset forth in claim 16 wherein the phospholipid vesicles targeted to thespecific locations in the body are labeled to identify such specificlocations.
 29. A method as set forth in claim 17, including the step of:labelling the monosaccharide containing lipid vesicles to identify thespecific locations of the tumor in the body.
 30. A method as set forthin claim 29 wherein the monosaccharide derivative of cholesterol has arange of approximately 0% to 20% by weight of the monosaccharides andthe remaining weight constituting the phospholipids.
 31. A method as setforth in claim 1, wherein radioactive labels are provided in thephospholipid vesicles for subsequent identification of the specificlocations of the tumor; and a chemical is chelated to such labels with aweak bond to provide for displacement by protein components in the bodywhen the phospholipid vesicles have targeted the specific locations ofthe tumor.
 32. A method as set forth in claim 6 wherein a drug orradionuclide is disposed within the micellular particles to be releasedat the specific locations of the tumor for treating the body at suchlocations.
 33. A method as set forth in claim 31 including the step of:chelating nitrilotriacetic acid to Indium-111 to provide a weak bondbetween the nitrilotriacetic acid and the Indium-111 and to provide fora displacement of the nitrilotriacetic acid by proteins in the body atthe specific location of the tumor.
 34. A method as set forth in claim 5wherein the phospholipid particles are labeled to facilitate theidentification of the specific locations of the tumor to which thephospholipid particles are targeted.
 35. The method of claims 6 or 32 inwhich distearoyl phosphatidylcholine and cholesterol are included insaid particles in a 2:1 molar ratio.